The present invention relates to the fields of molecular biology and microbiology. More specifically, this invention pertains to a novel gene encoding a putative efflux protein for solvents/antibiotics in Pseudomonas mendocina. 
Toxicity of aromatic solvents to microorganisms presents a major problem to work in the field of microbiology. Varied and poorly understood factors appear to influence solvent tolerance. Increasingly, attention has turned to genetic manipulation to create microbes that thrive in high concentrations of organic solvents. Understanding the mechanisms of solvent tolerance can be exploited in the future to generate microbes with enhanced biocatalytic potential.
One enzymatic pathway of increasing commercial interest controls toluene degradation. The first enzyme in the toluene degradation (TMO) pathway is toluene-4-monooxygenase (TMO; EC 1.14.13 and EC 1.18.1.3). Bacteria that possess the TMO pathway are useful for the degradation of toluene and other organics and are able to use these as their sole source of carbon (Wright et al., Appl. Environ. Microbiol. 60:235-242 (1994); Duetz et al., Appl. Environ. Microbiol. 60:2858-2863 (1994); Leahy et al., Appl. Environ. Microbiol. 62:825-833 (1996)). Bacteria that possess the TMO pathway are primarily restricted to the genus Pseudomonas. Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Pseudomonas mendocina are the most commonly utilized species.
Recently, various strains of Pseudomonas possessing the TMO pathway have been used to produce muconic acid from toluene via manipulation of growth conditions (U.S. Pat. No. 4,657,863; U.S. Pat. No. 4,968,612). Additionally, strains of Enterobacter with the ability to convert p-cresol to p-hydroxybenzoic acid (PHBA) have been isolated from soil (JP 05328981). Further, JP 05336980 and JP 05336979 disclose isolated strains of Pseudomonas putida with the ability to produce PHBA from p-cresol.
Although the above cited methods are useful for the production of PHBA, these methods are limited by the high cost and toxicity of the aromatic substrate, p-cresol. Furthermore, the above methods use an isolated wildtype organism that converts part of the p-cresol to PHBA while the remainder is further metabolized. The utility of these methods is limited by low yields and an inability to control further degradation of the desired product.
Previous studies indicated that cell growth and the activity of the enzymes in the metabolic toluene degradation pathway were inhibited in the presence of high concentrations of PHBA. Therefore, one problem to overcome is to develop a method of improving cell tolerance to high levels of PHBA or other aromatic solvents. During evolution, bacteria have developed a number of mechanisms which help to protect them from environmental toxins and various antibiotics. (Asako et al., Appl. Environ. Microbiol. 63:1428-1433 (1997); Aono et al., Appl. Environ. Microbiol. 60:4624-4626 (1994)). Many cytoplasmic membrane transport systems have been demonstrated to play an important role in bacteria by conferring resistance to toxic compounds. One of the most widespread is the active efflux of the toxic compounds from cells (Paulsen. et al., Microbiol. Rev. 60:575-608 (1996)).
Overexpression of an efflux system or its expression from a plasmid vector results in increased resistance of bacteria to a variety of toxic substances, while inactivation of an efflux system causes an increase in sensitivity to antibiotics and toxic substances (Li et al., J. Bacteriol. 180:2987-2991 (1998); Ramos. et al., J. Bacteriol. 180:3323-3329 (1998)). Such efflux systems are increasingly being recognized in a wide range of bacteria. Comparative amino acid sequence analysis of various transport proteins plus function assays has enabled the identification of a number of distinct families and super-families of transports.
The present invention provides a gene encoding a putative efflux protein for solvents or antibiotics in Pseudomonas mendocina. The invention further provides a Pseudomonas mendocina strain deficient in this gene that is unable to grow in the presence of chloramphenicol and, compared to the wildtype strain, grows slowly in the presence of high concentrations of PHBA.
The instant invention relates to isolated nucleic acid molecules encoding all or a substantial portion of a putative efflux protein. The isolated nucleic acid molecule encoding a putative efflux protein is selected from the group consisting of (a) an isolated nucleic acid molecule encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO:2; (b) an isolated nucleic acid molecule that hybridizes with the isolated nucleic acid molecule of (a) under the following hybridization conditions: 0.1xc3x97SSC, 0.1% SDS at 65xc2x0 C.; and (c) an isolated nucleic acid molecule that is complementary to (a) or (b). The nucleic acid molecules and corresponding polypeptides are contained in the accompanying Sequence Listing and described in the Brief Description of the Invention and the FIG. 2. More particularly, the invention is a 5.8 kb NotI nucleic acid molecule isolated from Psuedomonas mendocina KR-1 encoding a putative 4.5X gene and the accessory nucleic acid molecules for the expression of the gene as characterized by the restriction map of FIG. 2.
In another embodiment, the instant invention relates to chimeric genes encoding a putative efflux protein or to chimeric genes that comprise nucleic acid molecules as described above, the chimeric genes operably linked to suitable regulatory sequences, wherein expression of the chimeric genes results in altered levels of the encoded proteins in transformed host cells.
In a further embodiment, the instant invention concerns a recombinant host cell comprising in its genome a chimeric gene encoding a putative efflux protein as described above, operably linked to at least one suitable regulatory sequence, wherein expression of the chimeric gene results in production of altered levels of the putative efflux protein in the transformed host cell. The transformed host cells can be of eukaryotic or prokaryotic origin. A preferred embodiment uses E. coli as the host bacterium.
In an alternate embodiment, the present invention provides methods of obtaining a nucleic acid molecule encoding all or substantially all of the amino acid sequence encoding a putative efflux protein comprising either hybridization or primer-directed amplification methods known in the art and using the above described nucleic acid molecules. A primer-amplification-based method uses SEQ ID NO:1. The product of these methods is also part of the invention.
The invention further provides a method for the production of increased levels of PHBA comprising: (i) culturing a Pseudomonas strain in a medium containing an aromatic organic substrate, at least one suitable carbon source, and a nitrogen source, wherein the Pseudomonas strain comprises altered levels of a gene encoding a putative efflux protein, whereby PHBA accumulates; and (ii) recovering the PHBA.